[Show abstract][Hide abstract] ABSTRACT: Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia–reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the
Proceedings of the National Academy of Sciences 11/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the nuclear factor-kappaB (NF-kappaB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-kappaB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-kappaB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-kappaB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-kappaB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.
[Show abstract][Hide abstract] ABSTRACT: Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1(-/-) mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1(-/-)tnfr1(-/-) mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.
[Show abstract][Hide abstract] ABSTRACT: Members of the nuclear factor-[kgr]B (NF-[kgr]B) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-[kgr]B signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-[kgr]B activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-[kgr]B signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-[kgr]B target genes, and rapidly transformed neural stem cells[mdash]the cell of origin of ependymoma[mdash]to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeuti
[Show abstract][Hide abstract] ABSTRACT: The Nlrp3 inflammasome is critical for host immunity, but the mechanisms controlling its activation are enigmatic. In this study, we show that loss of FADD or caspase-8 in a RIP3-deficient background, but not RIP3 deficiency alone, hampered transcriptional priming and posttranslational activation of the canonical and noncanonical Nlrp3 inflammasome. Deletion of caspase-8 in the presence or absence of RIP3 inhibited caspase-1 and caspase-11 activation by Nlrp3 stimuli but not the Nlrc4 inflammasome. In addition, FADD deletion prevented caspase-8 maturation, positioning FADD upstream of caspase-8. Consequently, FADD- and caspase-8-deficient mice had impaired IL-1β production when challenged with LPS or infected with the enteropathogen Citrobacter rodentium. Thus, our results reveal FADD and caspase-8 as apical mediators of canonical and noncanonical Nlrp3 inflammasome priming and activation.
The Journal of Immunology 01/2014; 192(4). · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enhancement of cell death is a distinguishing feature of H1N1 influenza virus A/Puerto Rico/8/34 protein PB1-F2. Comparing the sequences (amino acids [a.a.] 61-87 using PB1-F2 a.a. numbering) of the PB1-F2-derived C-terminal peptides from influenza A viruses inducing high or low levels of cell death, we identified a unique I68, L69, and V70 motif in the A/Puerto Rico/8/34 PB1-F2 responsible for promotion of the peptide's cytotoxicity and permeabilization of the mitochondrial membrane. When administered to mice, a 27-mer PB1-F2-derived C-terminal peptide with this a.a. motif caused significantly greater weight loss and pulmonary inflammation than the peptide without it (due to I68T, L69Q, and V70G mutations). Similar to the wild-type peptide, A/Puerto Rico/8/34 elicited significantly higher levels of macrophages, neutrophils, and cytokines in the bronchoalveolar lavage fluid of mice than its mutant counterpart 7 days after infection. Additionally, infection of mice with A/Puerto Rico/8/34 significantly enhanced the levels of morphologically transformed epithelial and immune mononuclear cells recruited in the airways compared with the mutant virus. In the mouse bacterial superinfection model, both peptide and virus with the I68, L69, and V70 sequence accelerated development of pneumococcal pneumonia, as reflected by increased levels of viral and bacterial lung titers and by greater mortality. Here we provide evidence suggesting that the newly identified cytotoxic sequence I68, L69, and V70 of A/Puerto Rico/8/34 PB1-F2 contributes to the pathogenesis of both primary viral and secondary bacterial infections.
[Show abstract][Hide abstract] ABSTRACT: Caspase-8 or cellular FLICE-like inhibitor protein (cFLIP) deficiency leads to embryonic lethality in mice due to defects in endothelial tissues. Caspase-8(-/-) and receptor-interacting protein kinase-3 (RIPK3)(-/-), but not cFLIP(-/-) and RIPK3(-/-), double-knockout animals develop normally, indicating that caspase-8 antagonizes the lethal effects of RIPK3 during development. Here, we show that the acute deletion of caspase-8 in the gut of adult mice induces enterocyte death, disruption of tissue homeostasis, and inflammation, resulting in sepsis and mortality. Likewise, acute deletion of caspase-8 in a focal region of the skin induces local keratinocyte death, tissue disruption, and inflammation. Strikingly, RIPK3 ablation rescues both phenotypes. However, acute loss of cFLIP in the skin produces a similar phenotype that is not rescued by RIPK3 ablation. TNF neutralization protects from either acute loss of caspase-8 or cFLIP. These results demonstrate that caspase-8-mediated suppression of RIPK3-induced death is required not only during development but also for adult homeostasis. Furthermore, RIPK3-dependent inflammation is dispensable for the skin phenotype.
[Show abstract][Hide abstract] ABSTRACT: Regulated necrosis (RN) may result from cyclophilin (Cyp)D-mediated mitochondrial permeability transition (MPT) and receptor-interacting protein kinase (RIPK)1-mediated necroptosis, but it is currently unclear whether there is one common pathway in which CypD and RIPK1 act in or whether separate RN pathways exist. Here, we demonstrate that necroptosis in ischemia-reperfusion injury (IRI) in mice occurs as primary organ damage, independent of the immune system, and that mice deficient for RIPK3, the essential downstream partner of RIPK1 in necroptosis, are protected from IRI. Protection of RIPK3-knockout mice was significantly stronger than of CypD-deficient mice. Mechanistically, in vivo analysis of cisplatin-induced acute kidney injury and hyperacute TNF-shock models in mice suggested the distinctness of CypD-mediated MPT from RIPK1/RIPK3-mediated necroptosis. We, therefore, generated CypD-RIPK3 double-deficient mice that are viable and fertile without an overt phenotype and that survived prolonged IRI, which was lethal to each single knockout. Combined application of the RIPK1 inhibitor necrostatin-1 and the MPT inhibitor sanglifehrin A confirmed the results with mutant mice. The data demonstrate the pathophysiological coexistence and corelevance of two separate pathways of RN in IRI and suggest that combination therapy targeting distinct RN pathways can be beneficial in the treatment of ischemic injury.
Proceedings of the National Academy of Sciences 07/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Caspase-8, the initiator caspase of the death receptor pathway of apoptosis, its adapter molecule, FADD, required for caspase-8 activation, and cFLIPL, a caspase-8-like protein that lacks a catalytic site and blocks caspase-8-mediated apoptosis, are each essential for embryonic development. Animals deficient in any of these genes present with E10.5 embryonic lethality. Recent studies have shown that development in caspase-8-deficient mice is rescued by ablation of RIPK3, a kinase that promotes a form of programmed, necrotic cell death. Here, we show that FADD, RIPK3 double-knockout mice develop normally but that the lethal effects of cFLIP deletion are not rescued by RIPK3 deficiency. Remarkably, in mice lacking FADD, cFLIP, and RIPK3, embryonic development is normal. This can be explained by the convergence of two cell processes: the enzymatic activity of the FADD-caspase-8-cFLIPL complex blocks RIPK3-dependent signaling (including necrosis), whereas cFLIPL blocks RIPK3-independent apoptosis promoted by the FADD-caspase-8 complex.
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.
Molecular Medicine 02/2012; 18(1):577-86. · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An old puzzle in the field of cell death was solved recently: the mysterious embryonic lethality of animals deficient in caspase-8 or Fas-associated death domain (FADD), proteins involved in a pathway of apoptosis. This lethality is caused by a failure to develop the yolk sac vasculature rather than a lack of apoptosis. Remarkably, development is rescued by ablation of either of two receptor interacting serine-threonine kinases (RIPKs). Despite being well known cell killers, caspase-8 and FADD act together to block RIPK-mediated necrosis. To manifest this newly elucidated pro-survival function, FADD and caspase-8 depend on FLIP(Long), a catalytically inactive caspase-8 homolog. In this review, the mechanism by which RIPK necrotic death is inhibited by this trio is discussed, as well as how RIPKs might themselves mediate cell death.
Trends in cell biology 11/2011; 21(11):630-7. · 12.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Caspase-8, FADD, and FLIP orchestrate apoptosis in response to death receptor ligation. Mysteriously however, these proteins are also required for normal embryonic development and immune cell proliferation, an observation that has led to their implication in several nonapoptotic processes. While many scenarios have been proposed, recent genetic and biochemical evidence points to unregulated signaling by the receptor-interacting protein kinases-1 (RIPK1) and RIPK3 as the lethal defect in caspase-8-, FADD-, and FLIP-deficient animals and tissues. The RIPKs are known killers, being responsible for a nonapoptotic form of cell death with features similar to necrosis. However, the mechanism by which caspase-8, FADD, and FLIP prevent runaway RIPK activation is unknown, and the signals that trigger these events during development and immune cell activation remain at large. In this review, we will lay out the evidence as it now stands, reinterpreting earlier observations in light of new clues and considering where the investigation might lead.
[Show abstract][Hide abstract] ABSTRACT: The recent EMBO Molecular Medicine Workshop on Cell Death and Disease was held this past March in the picturesque Alpen ski-town of Obergurgl, Austria. Scientists working on diverse mechanisms and pathways of cell death convened to present and discuss their current research. Topics included not only cell death signalling pathways, their etiology in human disease, and potential avenues for therapeutic intervention, but also new approaches and perspectives for understanding the subtle mechanisms regulating cell fate.
EMBO Molecular Medicine 06/2011; 3(7):363-6. · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Caspase-8 has two opposing biological functions--it promotes cell death by triggering the extrinsic pathway of apoptosis, but also has a survival activity, as it is required for embryonic development, T-lymphocyte activation, and resistance to necrosis induced by tumour necrosis factor-α (TNF-α) and related family ligands. Here we show that development of caspase-8-deficient mice is completely rescued by ablation of receptor interacting protein kinase-3 (RIPK3). Adult animals lacking both caspase-8 and RIPK3 display a progressive lymphoaccumulative disease resembling that seen with defects in CD95 or CD95-ligand (also known as FAS and FASLG, respectively), and resist the lethal effects of CD95 ligation in vivo. We have found that caspase-8 prevents RIPK3-dependent necrosis without inducing apoptosis by functioning in a proteolytically active complex with FLICE-like inhibitory protein long (FLIP(L), also known as CFLAR), and this complex is required for the protective function.
[Show abstract][Hide abstract] ABSTRACT: Nitro-fatty acids (NO(2)-FAs) are emerging as a new class of cell signaling mediators. Because NO(2)-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO(2)-FAs in angiogenesis.
The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 μmol/L, both NO(2)-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO(2)-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1α (HIF-1α) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1α target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO(2)-FAs were slightly less potent inducers of EC migration and HIF-1α expression. Short hairpin RNA-mediated knockdown of HIF-1α attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO(2)-FAs.
Our data disclose a novel physiological role for NO(2)-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1α.
[Show abstract][Hide abstract] ABSTRACT: The death receptor ligands are involved in many physiological and pathological processes involving triggering of apoptosis, inflammation, proliferation, and activation. The expression of these molecules is reported to be tightly regulated at the transcriptional level. However, over the last few years, an increasing number of data demonstrated that the control of transcription is only one of the mechanisms that manage the expression of the death receptor ligands. Thus, this review is focused on posttranslational regulation of the three main members of this family, namely FasL, TNF-alpha, and TRAIL. We discuss here the importance of distribution, storage, and degranulation of these molecules, as well as their shedding by proteases on the control of death receptor ligands expression and activity.
Cellular and Molecular Life Sciences CMLS 03/2010; 67(10):1631-42. · 5.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4(+) T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation.
The Journal of Immunology 02/2010; 184(7):3487-94. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E(2) (PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE(2) in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.
Cell death and differentiation 10/2008; 15(12):1901-9. · 8.24 Impact Factor